Our laboratory has identified a sperm-specific dodecamer peptide sequence, designated as YLP12, vaccination with which causes a long-term reversible immunocontraceptive effect in female mice. In the present study, the effects of YLP12 DNA vaccine were examined. YLP12 36 bp cDNA was cloned into pVAX1 vector to prepare the DNA vaccine. Two additional vaccine constructs were made by in frame cloning of one and two CpG repeats in the YLP12-cDNA vaccine. Five groups of female mice were immunized intradermally by using gene gun with YLP12-cDNA, YLP12-cDNA-CpG, YLP12-cDNA-CpG-CpG, YLP12-cDNA mixed with exogenous synthetic CpG oligodeoxynucleotide (ODN), or vector DNA alone, respectively. Vaccination with all three constructs and the YLP12 vaccine mixed with exogenous ODN raised antibody response both in the sera as well as locally in the vaginal tract. There was no antibody response in the mice injected with the vector alone. In sera, the highest titers were obtained for the IgG class for all constructs and formulation followed by IgA class. In vaginal washings the highest titers were obtained for the IgA class followed by IgG class. Within the IgG class, the titers for the IgG2a subclass were significantly greater than the IgG1 subclass. Immunization with all constructs and formulation caused a significant (P < 0.05 to <0.001) reduction (20–43%) in fertility of female mice. The highest reductions were seen in mice immunized with YLP12-cDNA-CpG-CpG (two repeats) (43% reduction) and with the YLP12 vaccine administered with exogenous CpG ODN (42% reduction). T lymphocytes obtained from DNA-vaccinated mice showed clearly distinguished comparative RT-PCR analysis of cytokine mRNA expression for Th1 and Th2 immune responses compared to T lymphocytes obtained from control animals injected with vector DNA. Expression of both Th1 cytokines (IL-2 and IFN-gamma) and Th2 cytokines (IL-4 and IL-10) was enhanced after DNA vaccination as compared to controls, with a bias towards Th1 response. The immunocontraceptive effects were long-lasting observed up to 1.3 years of the observation period and increased with time. These novel findings indicate that the intradermal immunization with a sperm-specific DNA vaccine causes a long-term circulating and local immune response resulting in immunocontraceptive effects in female mice. Mol. Reprod. Dev. © 2006 Wiley-Liss, Inc.